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Dr.T.V.Rao MD
What is Typhoid FeverTyphoid fever, also known as typhoid, is a commonworldwide illness, transmitted by the ingestion of f...
Endemicity of Typhoid       Fever
Enteric FeversThe syndrome associatedwith enteric fevers areproduced only by a few ofthe SalmonellaSalmonella typhi mostim...
Bacteriology –Typhoid feverThe Genus Salmonellabelong toEnterobacteriaceaeFacultative anaerobeGram negative bacilliDisting...
Epidemiology of Typhoid FeverThis is a highly adapted, human-specific pathogen occurringmore frequently in underdeveloped ...
Antigenic structure of         SalmonellaTwo sets of antigensDetection by serotyping1 Somatic or 0 Antigens contain long c...
Diagnosis of Typhoid Fever (           CDC )Infection with typhoid orparatyphoid fever resultsin a very low-gradesepticemi...
WIDAL Test        land  Mark In Diagnosis            he Widal test is an old            serologic assay for detecting     ...
DiagnosisPhysician Still Plays the Key RoleBecause there is nodefinitive test for typhoid orparatyphoid fever, thediagnosi...
How we Diagnose Typhoid         FeverDiagnosis is made by any blood, bone marrow orstool cultures and with the Widal test(...
Blood Cultures in Typhoid         Fevers  Bacteremia occurs early  in the disease  Blood Cultures are  positive in1st week...
Identification of         SalmonellaSub cultures are doneafter overnightincubation at 370c,andsubcultures are doneon Mac C...
Salmonella on Mac Conkeys agar
Salmonella on XLD agar
Identifying Enteric         OrganismsIsolates which are Non lactose fermentingMotile, Indole positiveUrease negativeFermen...
Slide agglutination testsIn slide agglutinationtests a known serumand unknown cultureisolate is mixed,clumping occurs with...
Clot cultureClot cultures are moreproductive in yieldingbetter results in isolation.A blood after clotting, theclot is lys...
Bactek and Radiometric based  methods are in recent use                Bactek methods in                isolation of Salmo...
Widal Test      In 1896 Widal A professor of      pathology and internal      medicine at the University of      Paris (19...
Diagnosis of Enteric Fever                Widal testSerum agglutinins raise abruptly during the 2ndor 3rd weekThe widal te...
Significant Titers helps in        DiagnosisFollowing Titers of antibodiesagainst the antigens aresignificant when single ...
Widal test      Still a popular test ?The Widal test (Widal’s agglutination reaction) is routinelypractised for the serodi...
Limitations of Widal testClassically, a four-fold rise of antibody in pairedsera Widal test is considered diagnostic of ty...
Limitation of Widal TestThe Widal test is timeconsuming and oftentimes when diagnosis isreached it is too late tostart an ...
False Positive and Negative Reactions with WIDAL TestThe Widal test should be interpreted in the light of baselinetiters i...
WIDAL Test needs Quality               Control          Often Neglected ?Quality control of Widal tests is important: alab...
Advances in the RapidDiagnosis of Typhoid fever
Typhidot     Typhidot® a test kit that makes     use of 50 kD antigen to detect     specific IgM and IgG antibodies     to...
Typhidot is better than      Widal TestAnother variant of Typhidot®is Typhidot-M® and has shownthat inactivation of IgGrem...
Typhidot – Easier to Perform                The Typhidot offers an                additional advantage                amon...
ELISA Method                in  Diagnosis of Typhoid FeverA new technique of rapid screening for Salmonella bydipstick enz...
New Kits being tested in         fieldLaboratory confirmation of S. Typhi or S.Paratyphi as the etiologic agent will be es...
TUBEX®               TFTUBEX® TF is a 10 minutes semi-quantitative in vitrodiagnostic assay for detection of acute typhoid...
Principles of TUBEX TFTUBEX® TF is an inhibition binding assay. In short itdetects the presence of anti-O9 IgM antibodies ...
RT- PCR in TyphoidPCR amplification for thedetection of pathogens inbiological material isgenerally considered a rapidand ...
Search for Better Methods for Diagnosis of Typhoid FeverCurrently, alternative methods for biologicalmolecular analysis ar...
Several Emerging Methods       in DiagnosisCurrently, alternative methods for biologicalmolecular analysis are enzyme immu...
Molecular Immunology in Diagnosis of Typhoid FeverRecent advances inmolecular immunology haveled to the identification ofp...
Molecular Advances in Typhoid FeverFirst high-throughput functional analysis of every               Salmonella Typhi gene ...
Nano Technology in  Microbiology          Nanotechnology is an          emerging field that is          potentially changi...
Nano Technology in Diagnosis      of Typhoid FeverWith the development ofnanotechnology, variousnanoparticles and Nanoquan...
Newer Principles ofNanotechnology for TyphoidRecently, copper, silver, and gold-enhanced colloidal goldhave been reported ...
*References*NANOTECHNOLOGY AND THE DIAGNOSIS OF TYPHOID FEVERRAHUL MITRA Department of Banaras Hindu University, Varanasi-...
Created by Dr.T.V.Rao MD for          ‘e‘learning resources for Microbiologists in           Developing World             ...
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Typhoid Fever, Diagnosis

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Typhoid Fever, Diagnosis
Typhoid fever Newer Methods of Diagnosis.

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Typhoid Fever, Diagnosis

  1. 1. Dr.T.V.Rao MD
  2. 2. What is Typhoid FeverTyphoid fever, also known as typhoid, is a commonworldwide illness, transmitted by the ingestion of food orwater contaminated with the feces of an infected person, whichcontain the bacterium Salmonella enterica enterica serovar Typhi.The bacteria then perforate through the intestinal wall and arephagocytized by macrophages. The organism is a Gram-negative short bacillus that is motile due to its peritrichousflagella. The term"enteric fever" is a collective term thatrefers to typhoid and paratyphoid
  3. 3. Endemicity of Typhoid Fever
  4. 4. Enteric FeversThe syndrome associatedwith enteric fevers areproduced only by a few ofthe SalmonellaSalmonella typhi mostimportantSalmonella paratyphi A,B,C
  5. 5. Bacteriology –Typhoid feverThe Genus Salmonellabelong toEnterobacteriaceaeFacultative anaerobeGram negative bacilliDistinguished fromother bacteria byBiochemical andantigen structure
  6. 6. Epidemiology of Typhoid FeverThis is a highly adapted, human-specific pathogen occurringmore frequently in underdeveloped regions of the worldwhere overcrowding and poor sanitation are prevalent.According to the best global estimates, there are at least 16million new cases of typhoid fever each year, with 6, 00,000deaths (Ivanoff, 1995). Between 1 - 5% of patients with acutetyphoid infection have been reported to become chroniccarriers of the infection, depending on age, sex and treatmentregimen. Furthermore this chronic carrier state has also beenimplicated in causation of carcinoma of the gall bladder.
  7. 7. Antigenic structure of SalmonellaTwo sets of antigensDetection by serotyping1 Somatic or 0 Antigens contain long chainpolysaccharides ( LPS ) comprises of heat stablepolysaccharide commonly. 2 Flagellar or H Antigens are strongly immunogenicand induces antibody formation rapidly and in hightiters following infection or immunization. Theflagellar antigen is of a dual nature, occurring in oneof the two phases.
  8. 8. Diagnosis of Typhoid Fever ( CDC )Infection with typhoid orparatyphoid fever resultsin a very low-gradesepticemia. Blood cultureis usually positive in onlyhalf the cases. Stoolculture is not usuallypositive during the acutephase of the disease. Bone-marrow culture increases thediagnostic yield to about 80%of cases.
  9. 9. WIDAL Test land Mark In Diagnosis he Widal test is an old serologic assay for detecting IgM and IgG antibodies to the O and H antigens of Salmonella. The test is unreliable, but is widely used in developing countries because of its low cost. Newer serologic assays are somewhat more sensitive and specific than the Widal test, but are infrequently available.
  10. 10. DiagnosisPhysician Still Plays the Key RoleBecause there is nodefinitive test for typhoid orparatyphoid fever, thediagnosis often has to bemade clinically. Thecombination of a history ofbeing at risk for infectionand a gradual onset of feverthat increases in severityover several days shouldraise suspicion of typhoid orparatyphoid fever.
  11. 11. How we Diagnose Typhoid FeverDiagnosis is made by any blood, bone marrow orstool cultures and with the Widal test(demonstration of salmonella antibodies againstantigens O-somatic and H-flagellar ). Inepidemics and less wealthy countries, afterexcluding malaria, dysentery or pneumonia, atherapeutic trial time with chloramphenicol isgenerally undertaken while awaiting the resultsof Widal test and cultures of the blood and stool.
  12. 12. Blood Cultures in Typhoid Fevers Bacteremia occurs early in the disease Blood Cultures are positive in1st week in 90%2nd week in 75%3rd week in 60%4th week and later in 25%
  13. 13. Identification of SalmonellaSub cultures are doneafter overnightincubation at 370c,andsubcultures are doneon Mac Coneys agarSubcultures arerepeated upto 10 daysafter furtherincubation.
  14. 14. Salmonella on Mac Conkeys agar
  15. 15. Salmonella on XLD agar
  16. 16. Identifying Enteric OrganismsIsolates which are Non lactose fermentingMotile, Indole positiveUrease negativeFerment Glucose,Mannitol,MaltoseDonot ferment Lactose, SucroseTyphoid bacilli are anaerogenicSome of the Paratyphoid form acid and gasFurther identification done by slide agglutinationtests
  17. 17. Slide agglutination testsIn slide agglutinationtests a known serumand unknown cultureisolate is mixed,clumping occurs withinfew minutesCommercial sera areavailable for detectionof A, B,C1,C2,D, and E.
  18. 18. Clot cultureClot cultures are moreproductive in yieldingbetter results in isolation.A blood after clotting, theclot is lysed withStreptokinase ,butexpensive to perform indeveloping countries.
  19. 19. Bactek and Radiometric based methods are in recent use Bactek methods in isolation of Salmonella is a rapid and sensitive method in early diagnosis of Enteric fever. Many Microbiology Diagnostic Laboratories are upgrading to Bactek methods
  20. 20. Widal Test In 1896 Widal A professor of pathology and internal medicine at the University of Paris (1911–29), he developed a procedure for diagnosing typhoid fever based on the fact that antibodies in the blood of an infected individual cause the bacteria to bind together into clumps (the Widal reaction).
  21. 21. Diagnosis of Enteric Fever Widal testSerum agglutinins raise abruptly during the 2ndor 3rd weekThe widal test detects antibodies against O andH antigensTwo serum specimens obtained at intervals of 7 –10 days to read the raise of antibodies.Serial dilutions on unknown sera are tested against theantigens for respective SalmonellaFalse positives and False negative limits the utility of thetestThe interpretative criteria when single serum specimensare tested varyCross reactions limits the specificity
  22. 22. Significant Titers helps in DiagnosisFollowing Titers of antibodiesagainst the antigens aresignificant when single sampleis testedO > 1 in 160H > 1 in 320Testing a pairedsample for raise ofantibodies carries agreater significance
  23. 23. Widal test Still a popular test ?The Widal test (Widal’s agglutination reaction) is routinelypractised for the serodiagnosis of typhoid fever by most ofthe laboratories. Several workers have expressed doubtregarding the reliability of the test. Several factors havecontributed to this uncertainty. These include poorlystandardised antigens, the sharing of antigenic determinantswith other Salmonellae and the effects of immunisation withTAB vaccine. Another major problem relates to the difficultyof interpreting Widal test results in areas where Salmonellatyphi is endemic and where the antibody titres of the normalpopulation are often not known.
  24. 24. Limitations of Widal testClassically, a four-fold rise of antibody in pairedsera Widal test is considered diagnostic of typhoidfever. However, paired sera are often difficult toobtain and specific chemotherapy has to beinstituted on the basis of a single Widaltest. Furthermore, in areas where fever due toinfectious causes is a common occurrence thepossibility exists that false positive reactions mayoccur as a result of non-typhoid
  25. 25. Limitation of Widal TestThe Widal test is timeconsuming and oftentimes when diagnosis isreached it is too late tostart an antibioticregimen.In spite of severallimitation manyPhysicians depend onWidal Test
  26. 26. False Positive and Negative Reactions with WIDAL TestThe Widal test should be interpreted in the light of baselinetiters in a healthy local population. This is especially importantwhen there is a high local prevalence of non-typhoidsalmonellosis.4 The Widal test may be falsely positive inpatients who have had previous vaccination or infection with Styphi.7 RaisedWidal titers have also been reported in association with thedysgammaglobulinaemia of chronic active hepatitis and otherautoimmune diseases.64 8 9 False negative results may beassociated with early treatment, with "hidden organisms" inbone and joints, and with relapses of typhoid fever.Occasionally the infecting strains are poorly immunogenic.
  27. 27. WIDAL Test needs Quality Control Often Neglected ?Quality control of Widal tests is important: alaboratory which consistently produces poorresults in an external quality control programmeshould discontinue the test until technicalproblems are solved. False positive results maybe due to faulty technique or to poor quality ofthe antigen suspension. There is conflictingevidence as to the relative importance of somaticand flagellar agglutinin titers for the diagnosis oftyphoid fever
  28. 28. Advances in the RapidDiagnosis of Typhoid fever
  29. 29. Typhidot Typhidot® a test kit that makes use of 50 kD antigen to detect specific IgM and IgG antibodies to S. typhi (Ismail et al., 1991). It has undergone full - scale multinational clinical evaluation of its diagnostic value (Lu-Fong et al., 1999; Jackson et al., 1995; Choo et al., 1997). This dot EIA test offers simplicity, speed, specificity (75%), economy, early diagnosis, sensitivity (95%) and high negative and positive predictive values
  30. 30. Typhidot is better than Widal TestAnother variant of Typhidot®is Typhidot-M® and has shownthat inactivation of IgGremoves competitive bindingand allows access of theantigen to the specific IgMwhen it is present. Evaluationof Typhidot® and Typhidot-M®in clinical settings showedthat they performed better thanthe Widal test and the culturemethod (Bhutta and Mansurali,1999).
  31. 31. Typhidot – Easier to Perform The Typhidot offers an additional advantage among second- line serologic diagnostic tests for typhoid fever in that the test strips do not require an ELISA reader for evaluation. Also, only minimal operator training is required
  32. 32. ELISA Method in Diagnosis of Typhoid FeverA new technique of rapid screening for Salmonella bydipstick enzyme-linked immuno sorbent assay (ELISA)has been shown to be sensitive, specific, rapid andreproducible for detection of Salmonella directly fromstool. Stool samples are mixed with buffer Salmonellainteraction solution. A dipstick is placed into the mixtureand incubated at room temperature.Results of the tests are available in 20 minutes. Early publishedresults from different studies show a Sensitivity rate of 99% andspecificity of 98%. The dipstick kit is very useful and acceptable toboth patients and health-care providers because of the followingreasons
  33. 33. New Kits being tested in fieldLaboratory confirmation of S. Typhi or S.Paratyphi as the etiologic agent will be essentialto distinguish typhoid/paratyphoid fromnumerous other causes of acute febrile illness. Arapid diagnostic test (Tubex TF, IDL Biotech,Bromma, Sweden) can detect typhoid (but notparatyphoid) antibody in patient serum. In fieldtrials, the Tubex TF kit had a sensitivity of 60–78% and a specificity of 58–89%
  34. 34. TUBEX® TFTUBEX® TF is a 10 minutes semi-quantitative in vitrodiagnostic assay for detection of acute typhoid fever. Itspecifically detects the presence of IgM antibodies to theS. typhi O9 lipopolysaccharide antigen. This antigen ishighly specific to S. typhi and other Salmonella serogroupD bacteria by its extremely rare sugar (a-D-tyvelose). IgManti-O9 antibodies are normally not present in healthyindividuals, and thus TUBEX® TF is highly specific. Apositive TUBEX® TF result, together with typical clinicalsymptoms of typhoid fever, is highly suggestive of atyphoid infection.
  35. 35. Principles of TUBEX TFTUBEX® TF is an inhibition binding assay. In short itdetects the presence of anti-O9 IgM antibodies in patientserum by assessing the ability to inhibit a reactionbetween two colored and antigen/antibody-coatedreagents. TUBEX® TF is a semi-quantitative assay, andthus the level of inhibition is proportional to theconcentration of anti-O9 antibodies in the sample. Theseparation is performed in one step by magnetic force;where after the result is read visually and scored against aprovided color scale.
  36. 36. RT- PCR in TyphoidPCR amplification for thedetection of pathogens inbiological material isgenerally considered a rapidand informative diagnostictechnique..Several experimentalmethods for PCR methodsin progress.Needs greater validation
  37. 37. Search for Better Methods for Diagnosis of Typhoid FeverCurrently, alternative methods for biologicalmolecular analysis are enzyme immunoassay ,surface plasmo resonance , an electrochemicalimmunoassay . In particular, the use ofelectrochemical immunoassay has attractedconsiderable interest for S.typhi determinationbecause of its inherent simplicity, highsensitivity, inexpensive instrumentation, andminiaturization
  38. 38. Several Emerging Methods in DiagnosisCurrently, alternative methods for biologicalmolecular analysis are enzyme immunoassay ,surface plasmo resonance , an electrochemicalimmunoassay . In particular, the use ofelectrochemical immunoassay has attractedconsiderable interest for S.typhi determinationbecause of its inherent simplicity, highsensitivity, inexpensive instrumentation, andminiaturization
  39. 39. Molecular Immunology in Diagnosis of Typhoid FeverRecent advances inmolecular immunology haveled to the identification ofpotentially more sensitiveand specific markers in theblood and urine of patientswith typhoid fever andenabled the manufacture ofpractical and inexpensivekits for their detection.
  40. 40. Molecular Advances in Typhoid FeverFirst high-throughput functional analysis of every Salmonella Typhi gene The team were able to look at almost all the genes in S. Typhi and showed that it needs only 356 genes for survival: 4162 genes were not essential. Knowing which genes are essential to the survival of pathogens, researchers can seek treatments to target those genes.
  41. 41. Nano Technology in Microbiology Nanotechnology is an emerging field that is potentially changing the way we treat and diagnose diseases. The metal-enhanced colloidal gold has not been previously applied to the detection of bacterial cells in real samples
  42. 42. Nano Technology in Diagnosis of Typhoid FeverWith the development ofnanotechnology, variousnanoparticles and Nanoquantum dots have beenused as labels to enhancethe sensitivity of theelectrochemicalimmunoassay technique
  43. 43. Newer Principles ofNanotechnology for TyphoidRecently, copper, silver, and gold-enhanced colloidal goldhave been reported for immunoglobin G (IgG)determination, which is the model of electrochemicalimmunoassay with low detection limits ranged from 1.0ng/mL to 0.25 pg/mL . The metal-enhanced colloidalgold electrochemical stripping metalloimmunoassaycombines the high sensitivity of stripping metal analysiswith the remarkable signal amplification resulting fromthe catalytic precipitation of metals onto the goldnanoparticles
  44. 44. *References*NANOTECHNOLOGY AND THE DIAGNOSIS OF TYPHOID FEVERRAHUL MITRA Department of Banaras Hindu University, Varanasi- 221005,INDIA, Digest Journal of Nanomaterials and Bio structures, Vol. 4, No. 1,March 2009, p. 109-111CDC Typhoid Fever General Information
  45. 45. Created by Dr.T.V.Rao MD for ‘e‘learning resources for Microbiologists in Developing World Email doctortvrao@gmail.com

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